1. Field Of the Invention
The present invention relates to a chamber for freeze-drying by cryosorption, wherein cooling is effected by liquid nitrogen in a Dewar vessel.
2. Description of Related Art
In order to better present fine structures or features of a biological specimen under an optical or electron microscope, especially in the case of histochemical applications, freeze-drying (hereinafter referred to as "FD") of a biological specimen is becoming more widely used. Typically, when a biological specimen is freeze-dried, the specimens are, without any pretreatment, frozen extremely rapidly ("cryofixation") and subsequently dried, preferably in a vacuum at temperatures of around -80.degree. C. (in this connection, c.f. inter alia H. D. Coulter and L. Terracio, Anat. Rec. 187, 477-494, 1960; V. Hanzon and L. H. Hermodsson, Ultrastruct. Res. 4, 332-348, 1960; F. D. Ingram and M. J. Ingram, Scanning Electron Microscopoy, IV, 147-160, 1980; J. G. Linner et al., J. Histochem. Cytochem. 34, 1123-1135, 1986; J. D. Mellor, Fundamentals of Freeze-Drying, Academic Press, London, 1978; H. T. Meryman in H. T. Meryman, Editor, Cryobiology, Academic Press, Sn. 609-663, 1966; K. Neumann, Grundri.beta. der Gefriertrocknung (Outline of Freeze Drying), Musterschmidt Verlag, Gottingen, 1952; and references and publications cited therein).
According to the cited prior art, in most instances, freeze-drying of biological specimens is conducted in systems wherein the vacuum is generated by a two-stage pump system (a rotary prevacuum pump with a diffusion high vacuum pump) or a single stage pump system (a turbomolecular pump), and the external cooling of the biological specimens and condensation surfaces is carried out with liquid nitrogen (hereinafter referred to as "LN.sub.2 "). In addition to the considerable expense of the apparatus and equipment necessary to conduct the freeze-drying of biological specimens in this manner, very high operating costs are also incurred because the majority of commercially available or laboratory systems use over 1 liter of LN.sub.2 per hour and also because even FD of small specimens having a diameter of less than about 1 mm requires an average of approximately 14 days. Moreover, a majority of these systems generate high levels of noise owing to the continuously running rotary pumps and therefore does not permit operation of such FD systems in common areas. Another drawback of these systems stems from the especially high labor costs associated with the need to continuously and frequently refill the LN.sub.2. Attempts at automating the chore of refilling LN.sub.2 have the effect of increasing the capital expenditure to a considerable extent.
Finally, a substantial additional disadvantage of these systems resides in the fact that all rotary pumps and diffusion pumps liberate oil vapors, which are deposited on the cold object surfaces and, as a result of this, alter such surfaces artifactually.
Accordingly, repeated attempts were made to use cryosorption systems for the generation of a clean oil-free high vacuum (in this connection, c.f., inter alia German Patent DE 27 39 796 as well as L. Edelmann, Microscopy, Vienna, 35, 31-36, 1979; L. Edelmann, Scanning Electron Microscopy, IV, 1377-1356, 1986; and further literature references cited therein). The system developed and used by Edelmann comprises a divisible cylindrical container with a volume of approximately 0.5 liter, which is connected in a vacuum-tight manner to a tube, the free end of which is equipped with a valve and a hose connection. In the lower part of the container there is a molecular screen as a drying agent. A thermostatically heatable tray to receive the frozen specimens is secured in the upper part of the container. After filling of the molecular screen and loading of the specimen tray with the frozen specimens, both parts are screwed to one another in a vacuum-tight manner. Thereafter, the container is introduced into an LN.sub.2 -filled Dewar vessel. At this point, all of the wall surfaces of the container are in direct contact with LN.sub.2 (temperature -196.degree. C.), and the molecular screen is also cooled and adsorbs most of the gas molecules (O2, N.sub.2, CO.sub.2, H.sub.2 O) present in the container. Depending upon whether and to what extent the noble gases are or are not drawn off by a prevacuum pump, the result is a vacuum between 10.sup.-3 and 10.sup.-5 torr, which is entirely sufficient for an efficient FD, relative to the given mean free path length of the H.sub.2 O molecules. The situation is additionally improved in that H.sub.2 O is permanently deposited on the chamber walls directly cooled by LN.sub.2, and in that the decisive water vapor partial pressure of this arrangement becomes almost immeasurably small as a result of this. A particular advantage of the described arrangement finally resides in that the LN.sub.2 consumption of the system is below 1 liter of LN.sub.2 per day. Accordingly, drying over 14 days in a 35 liter Dewar vessel can be performed without LN.sub.2 refilling and without noise.
As against the cited advantages of cryosorption FD according to Edelmann, there are disadvantages which oppose its routine use. These include in the first instance the complicated loading of the chamber with the molecular screen and the frozen specimens. At low temperatures and when the container is in the first instance open, the molecular screen immediately absorbs gases and this leads to a loss of cryosorption capacity. The sealing of the two-part chamber is very difficult and requires high forces, since only hard sealing rings, preferably metal seals, are suitable in the temperature range &lt;-180.degree. C. It is very difficult to secure the correct position of the specimens in the hollows of the specimen tray during this procedure. Finally, any visual monitoring and UV polymerization in this system is impossible. All cited problems are likewise applicable, in some cases to an intensified extent, to the removal of the specimens which have become extremely hygroscopic as a result of the FD.
In view of the indisputable advantages of a cryosorption system for FD, an object of the present invention is to provide an arrangement which fully exploits the advantages of the Edelmann container without its disadvantages, and is thus competent for routine use without restrictions.
According to the present invention, this object is achieved as a refinement or improvement of the Edelmann concept by providing a chamber for freeze-drying by cryosorption, having a cooling effected by liquid nitrogen, which chamber is situated in a Dewar vessel and cools a body having good thermal conductivity, wherein the chamber comprises
a cylindrical metal outer walling and is primarily situated within the Dewar vessel, preferably within the neck of the Dewar vessel, the lower edge of the cylindrical metal outer walling of the container being connected in a vacuum-tight manner to a lower edge of a rotary component which is preferably rotationally symmetric, PA1 the rotary component having a planar bottom exhibiting a contact surface, which planar bottom corresponds with the likewise preferably planar complementary surface on top of a cylindrical body around which liquid nitrogen flows and which is cooled by this cylindrical body, PA1 the rotary component having a chamber for receiving the drying agent for cryosorption, for example a molecular screen, as well as a connection to the drying chamber, PA1 an upper edge of the cylindrical metal walling optionally having a ring which is connected thereto in a vacuum-tight manner situated outside the Dewar vessel and therefore approximately at ambient temperature. To this end, the arrangement according to the invention exhibits a cooling which takes place in a known manner on the floor surface of a container, such that this floor surface corresponds with a complementarily designed surface on the top of a LN.sub.2 -cooled solid body which is situated in a Dewar vessel, and the height of the LN.sub.2 level in the Dewar vessel does not exert any significant influence on the temperature of the contact surface because of the material cross section and of the thermal conductivity of this solid body, which temperature as a rule differs from the LN.sub.2 temperature (-196.degree. C.) by at most 20.degree. C.
The floor of the FD chamber is preferably a part of a rotary component of a metal of good thermal conductivity (e.g., brass or aluminum), which exhibits a chamber to receive a drying agent (e.g., "zeolite" molecular screen) as well as, at its top, a depression for a tray for receiving the frozen specimens. The chamber for the drying agent exhibits at least one connection to the freeze-drying chamber and, if required, in addition an opening, which opening can be sealed in a vacuum-tight manner, for the filling and exchange of the drying agent. The side walls of the chamber are formed by a preferably cylindrical sleeve of a thinmetal sheet of poor thermal conductivity (e.g., fine steel sheet in a thickness of approximately 0.2 to 1 mm and a height &gt;100 mm). The cylindrical sleeve is connected at its lower end in a vacuum-tight manner to the aforementioned rotary component, and at its upper edge, likewise in a vacuum-tight manner, to a metal ring having good thermal conductivity. The position of the chamber and the height of the side walls are dimensioned so that at least the topmost portion of the chamber with the metal sealing ring projects from the neck of the Dewar vessel.
Accordingly, under normal operating conditions during FD, the topmost portion of the chamber is at a temperature which is above the dew point of normal ambient air. In this way, it is possible to seal off the sealing cover, over the opening in the upper sealing ring of the chamber, with a commercially available O-ring. Identical conditions apply to a normal vacuum connection (e.g., standard flange), which can preferably be disposed in that region of the chamber which projects out of the Dewar vessel, for example again at the upper sealing ring, and connects the FD chamber space to the ambient air. It is possible to connect to it any commercially available vacuum components (e.g., vacuum meter, valves, hose connections for prevacuum pumps, etc.).
To receive and thermostatically heat the frozen specimens, it is possible to provide a tray in the depression at the top of the floor rotary component of the chamber, which tray exhibits a heating cartridge and a temperature sensor for the thermostatic heating as well as, at its top, at least one hollow to receive the frozen specimens. The mounting of this tray on the floor rotary component takes place preferably by point or line overlays, in such a manner that only a minimal amount of thermal contact results between the deep-frozen rotary component (&lt;-176.degree. C.) and the specimen tray, which tray is normally heated to approximately -80.degree. C. for FD.
An additional feature of the present invention resides in the floor rotary component upon which additional cooling surfaces may be attached and thereby increase the binding of H.sub.2 O. For the permanent assurance of a thermal contact, that cannot be influenced by manipulation forces, between the floor surface of the chamber and the complementary solid body surface in the Dewar vessel, the chamber may be screwed together with the Dewar vessel or to a mounting component fastened to the Dewar vessel. Moreover, according to the present invention, a screw connection component which is pretensioned by spring elements can compensate for any length changes of the cooled parts.
A further feature of the present invention is that valves effect a blocking of the chamber with the drying agent in the floor component, as well as an opening of a connection to the interior of the Dewar vessel and thus a flooding of the FD chamber with dry cold nitrogen gas (hereinafter referred to as "GN.sub.2 "). The first valve, can at the same time, be designed as an overpressure valve and thus protect the user against a spontaneous release of adsorbed gases when after lengthy dry periods and as the chamber warms up after vaporization of the last LN.sub.2 residues from the Dewar vessel, the absence of special precautions could lead to an explosion of the floor part.
A further feature of the present invention is that additional vacuum connections, as well as a further valve with a downstream hose connection for the preevacuation of the chamber space for the removal of a major part of the noble gases are provided. Moreover, the cover for sealing the upper chamber opening may comprise a transparent and/or UV-transmitting material. Further, the upper sealing ring and the valves are provided with at least one heating cartridge and a temperature sensor, which permit a thermostatic heating of these elements as protection against a disturbing cooling during a relatively long period of flooding of the chamber with dry cold GN.sub.2.